Determination of Porphyromonas antimicrobial resistance mechanisms by Tn-Seq

通过 Tn-Seq 确定卟啉单胞菌耐药机制

• 批准号: 8628666
• 负责人: BRIAN Andrew KLEIN
• 金额: $ 3.92万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8628666
• 关键词: Affect; Anaerobic Bacteria; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Bacteria; Basic Science; Biological Models; Caring; Clinical; Communicable Diseases; Complement; DNA; Defect; Defensins; Dental; Dental caries; Development; Economic Burden; Educational Status; Exhibits; Exposure to; Future; Gene Targeting; Genes; Genetic; Genome; Gentamicins; Goals; Gram-Negative Anaerobic Bacteria; Growth; Host Defense; Hot Spot; Human; In Vitro; Individual; Infection; Lead; Learning; Libraries; Light; Link; Massive Parallel Sequencing; Methodology; Methods; Metronidazole; Metronidazole resistance; Microbial Biofilms; Mus; Mutagenesis; Mutation; Nitroimidazoles; Oral; Organism; Pathogenesis; Pathway interactions; Patients; Periodontal Diseases; Periodontitis; Pigmentation physiologic function; Play; Population; Porphyromonas; Porphyromonas gingivalis; Positioning Attribute; Pre-Par; Process; Relapse; Relative (related person); Reporting; Research; Resistance; Role; Running; Sampling; Site; Socioeconomic Status; Surface; System; Techniques; Technology; Time; Validation; Virulence; Virulence Factors; Virulent; Work; antimicrobial; antimicrobial drug; antimicrobial peptide; base; care burden; fitness; genetic manipulation; insight; killings; mouse model; mutant; oral bacteria; pathogen; peptide B; pressure; resistance mechanism; tool; treatment strategy

DESCRIPTION (provided by applicant): Porphyromonas gingivalis, an anaerobic, Gram-negative bacterium, is an important organism in the pathogenesis of periodontitis. Antibiotics are used in the treatment of severe periodontitis and recently growing resistance to antibiotics in clinical P.gingivalis isolates has been reported. Metronidazole is a nitroimidazole with activity against many anaerobic bacteria, including P.gingivalis. Resistance to metronidazole has now been reported as high as 22%, but little is known about the mechanisms of resistance. In this proposal, I will use transposon mutagenesis to identify genes and gene networks involved in the development of metronidazole resistance. In my preliminary work, I have created two transposon mutant libraries in different strains of P.gingivalis using a Mariner-based transposon system. This transposon system has advantages compared to prior transposon libraries in P.gingivalis due to ability of Mariner to insert randomly as to create highly saturated libraries with insertions throughout the genome. Using this library, I have already identified previously unrecognized pigmentation mutants of P.gingivalis. Interestingly, some of these pigmentation-defective Pg mutants are resistant to metronidazole. In this proposal, I will first establish the rle of pigmentation in resistance to metronidazole by performing complementation and characterization of the pigmentation mutants I have isolated. The transposon I used is adapted for use with massively-parallel sequencing to identify the relative fitness of mutants in the libray with a technique called Tn-seq. To uncover additional non-pigment related genes involved in metronidazole resistance, I will perform a screen of the entire library using Tn-seq to identify mutants that are either enriched or eliminated on exposure to different concentrations of metronidazole. Finally, I will perform clinical sampling for P.gingivalis from patients with periodontal disease and employ targeted gene sequencing of metronidazole resistance genes identified in Aims 1 and 2, to determine whether mutations associated with in vitro resistance to metronidazole are found in patients with clinically resistant isolates of P.gingivalis. This projec is expected to shed new light on mechanisms of P.gingivalis resistance to an important antimicrobial agent. In the process I will be developing new tools and learning techniques that have the potential to be applied to multiple aspects of P.gingivalis virulence and that will prepar me for a future in oral microbiological research.

描述（由申请人提供）：牙龈卟啉单胞菌是一种厌氧的革兰氏阴性细菌，是牙周炎发病机制中的重要微生物。抗生素被用于治疗严重的牙周炎，并且最近已经报道了临床牙龈卟啉单胞菌分离株对抗生素的耐药性增加。甲硝唑是一种硝基咪唑，对许多厌氧菌具有活性，包括牙龈卟啉单胞菌。据报道，对甲硝唑的耐药率高达22%，但对耐药机制知之甚少。在这个提议中，我将使用转座子诱变来鉴定参与甲硝唑耐药性发展的基因和基因网络。在我的初步工作中，我已经使用基于Mariner的转座子系统在不同菌株的牙龈卟啉单胞菌中创建了两个转座子突变体文库。与牙龈卟啉单胞菌中的现有转座子文库相比，该转座子系统具有优势，这是由于Mariner随机插入的能力，从而产生在整个基因组中具有插入的高度饱和文库。使用这个库，我已经确定了以前未被识别的色素沉着牙龈卟啉单胞菌突变体。有趣的是，这些色素缺陷型Pg突变体中的一些对甲硝唑具有耐药性。在这个建议中，我将首先通过对我分离的色素沉着突变体进行互补和表征来建立色素沉着在甲硝唑抗性中的作用。我使用的转座子适合与平行测序一起使用，以通过一种称为Tn-seq的技术来鉴定库中突变体的相对适合度。为了揭示与甲硝唑耐药性相关的其他非色素相关基因，我将使用Tn-seq对整个文库进行筛选，以鉴定暴露于不同浓度甲硝唑时富集或消除的突变体。最后，我将对牙周病患者的牙龈卟啉单胞菌进行临床采样，并对目标1和2中确定的甲硝唑耐药基因进行靶向基因测序，以确定是否在临床耐药牙龈卟啉单胞菌分离株患者中发现与甲硝唑体外耐药相关的突变。本研究有望为牙龈卟啉单胞菌耐药机制的研究提供新的思路。在此过程中，我将开发新的工具和学习技术，这些工具和技术有可能应用于牙龈卟啉单胞菌毒力的多个方面，并为我未来的口腔微生物研究做好准备。